Accelerated metabolite identification by "extraction-NMR".

Examples of the use of extraction-NMR, an efficient and rapid method to obtain structural information on metabolites without prior separation, are described. Crude ethyl acetate extracts of in vitro microsomal incubations were analysed by NMR spectroscopy. The region downfield of 5.5 ppm in the proton spectra of these crude extracts was found to be essentially clear of endogenous resonances. As a consequence, sites of aromatic hydroxylation can often be determined without prior separation of the crude extracts. Sites of metabolism close to the aromatic region may also be accessible via two-dimensional (2D) homonuclear experiments (e.g. COSY, NOESY, TOCSY). One-dimensional (1D) and 2D fluorine experiments also can provide additional information on the structure of metabolites. Depending on the complexity of the aromatic region of the parent compound, signal overlap and the relative abundance of the individual components, extraction-NMR has the potential to provide information for unambiguous structure elucidation of two or three major metabolites. Should extraction-NMR produce inconclusive results, i.e. too many metabolites are present or metabolism occurred exclusively on aliphatic regions, it is possible to re-use the extraction-NMR sample and proceed with traditional methods of analysis.